青枯雷尔氏菌胞外多糖合成缺失突变株构建及其生物学特性

Construction and characterization of extracellular polysaccharide deletion mutant of Ralstonia solanacearum

摘要:

[目的]由青枯雷尔氏菌（Ralstonia solanacearum）引起的植物青枯病是一种毁灭性土传病害。胞外多糖（extracellular polysaccharides，EPS）是青枯雷尔氏菌关键的致病因子之一。通过构建胞外多糖缺失突变株，研究胞外多糖在青枯病致病中的作用。[方法]从青枯雷尔氏菌FJAT-91的基因组中克隆出胞外多糖合成结构基因epsD同源臂，克隆至自杀性质粒pK18mobsacB，再将庆大霉素抗性基因（Gm）插入同源臂中间，获得重组质粒pK18-epsD。将重组质粒转化至青枯雷尔氏菌FJAT-91感受态细胞中，通过同源重组敲除epsD基因，获得EPS合成缺失的突变株FJAT-91ΔepsD。研究突变株与野生菌株在菌落形态、胞外多糖合成、运动能力、定殖能力的差异性。[结果]突变菌株FJAT-91ΔepsD与出发菌株FJAT-91相比：胞外多糖产量显著减少，生长较慢；泳动能力（swimming motility）和群集运动能力（swarming motility）显著降低；在番茄苗根部和茎部的定殖能力显著降低；弱化指数（AI）为0.905，鉴定为无致病力菌株。[结论]胞外多糖在青枯雷尔氏菌的致病中起着关键的作用，本课题研究成果为开发植物疫苗提供了优良的材料与研究基础。

Abstract:

[Objective] Bacterial wilt is a soil-borne disease caused by Ralstonia solanacearum. Extracellular polysaccharides are one of the key pathogenic factors of R. solanacearum. The physiological functions of extracellular polysaccharides on the pathogenesis of bacterial wilt were studied by constructing a mutant with synthesis of extracellular polysaccharides deficiency. [Methods] First, we cloned the homologous arm of epsD from the genome of R. solanacearum FJAT-91, then inserted into suicide plasmid pK18mobsacB. Second, the Gm gene was inserted into homologous arm to obtain recombinant plasmid pK18-epsD. Third, the recombinant plasmid was transformed into R. solanacearum FJAT-91 competent cells. The epsD gene deletion mutant was constructed by homologous recombination. Finally, we detected the differences of the biological characteristics of mutant strains and wild-type strains. [Results] Compared with the wild-type strains, the mutant strains showed different features. The mutant showed: grew slowly and decreased the yield of extracellular polysaccharides; significantly reduced the abilities of swimming motility and swarming motility; significantly decreased the colonization ability in roots and stems of tomato; had the Attenuation Index (AI) with 0.905 and the avirulence to tomato which indicated a nonpathogenic strain. [Conclusion] These results suggested that extracellular polysaccharides played the key role in pathogenicity of R. solanacearum. This research provides excellent materials and research foundation for the development of plant vaccines.